Summary Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ cycling proteins of sinoatrial node cells (SANC) is higher than in left ventricular myocytes (LVC) and is crucial for SANC basal normal automaticity. We hypothesized that: (1) the level of Ca2+ cycling protein phosphorylation expression and abundance of Ser/Thr phosphoprotein phosphatases (PP) PP1 and PP2A or endogenous PP1 inhibitors (Inhibitor-1 (I-1), kinase C-enhanced PP1 inhibitor (KEPI) and Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32)) differ in SANC and LVC, and (2) that action potential (AP) firing rate of SANC is linked to PP activity. PP1 expression was equivalent in SANC and LVC; PP2A transcript expression in LVC exceeded that in SANC by about 4-fold. Transcript expression of KEPI in LVC exceeded that in SANC, I-1 and DARPP-32 expression was similar in both cell types. PP1 protein abundance (Western blot) in SANC averaged 50% of that in LVC, whereas average DARPP-32 abundance in SANC was 2-fold higher than in LVC; in contrast to other PP1 inhibitors, KEPI protein was 4-fold higher in LVC than in SANC. Inhibition of both PP1 and PP2A activities by Calyculin A (CyA) in intact single SANC significantly (by 2-3-fold) increased phospholamban (PLB) phosphorylation at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites. In freshly isolated single rabbit SANC, CyA (100 nmol/L) decreased the spontaneous SANC cycle length and increased the average spontaneous SANC firing rate (perforated patch-clamp technique) by30%. CyA substantially increased diastolic local Ca2+ release (LCR) number and size (confocal line-scan imaging) and reduced the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR). The CyA-induced increase in LCR parameters was accompanied by an increase in RyR phosphorylation at Ser2809 site, concurrent augmentation of L-type Ca2+ current amplitude and elevation of sarcoplasmic reticulum Ca2+ load. The selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on SANC spontaneous AP firing, LCR parameters or PLB phosphorylation. In permeabilized SANC CyA markedly increased LCR number and size and purified PP1 suppressed LCRs, while neither okadaic acid nor purified PP2A affected LCR characteristics. A numerical model, fine-tuned by experimentally observed changes in ICaL, PLB phosphorylation and changes in SR Ca2+ pumping produced by PP inhibition in SANC quantitatively predicted changes in SANC AP firing rate in response to PP inhibition. Thus, expression patterns of PPs and PP1 inhibitors in SANC differ substantially from those in LVC, and basal PP activity modulates the basal spontaneous firing rate of SANC via suppressive effects on SR Ca2+ cycling protein phosphorylation, SR-generated LCRs and L-type Ca2+ current.